Development of an electrochemical dopamine sensor for monitoring the neurotransmitter release of human midbrain organoids on-chip

Abstract

The neurotransmitter dopamine is not only known as the happiness hormone but also as an essential biomarker for the second most common neurogenerative disease, with9.4million patients world wide: Parkinson’s disease. Until now, the treatment of Parkinson’s disease remained symptomatic due to the lack of neuroprotective and disease-altering treatment strategies as well as suitable disease models. The gold standard animal models suffer from a high failure rate in clinical trials due to inept recapitulation of the highly heterogeneous disease. An alternative to conventional animal models is organ-on-chip technology that employs three-dimensional, self-structured, miniaturized tissues generated from patient- specific cells to mimic tissues in vitro. Static cultivation of organoids suffers mainly from an insufficient simulation of the necessary environment due to a lack of biophysical stimuli, which is, in contrast, addressed by the dynamic cultivation using microchannels inside microfluidic platforms. An advantage of organ-on-chips with organoids is the ability to employ sensing approaches for increasing analytical accessibility. In the case of Parkinson’s specific organoid-on-chip models, the integration of non-invasive dopamine sensing strategies enables the monitoring of the neurotransmitter release. Limitations of dopamine detection are the highly reactive and unstable behavior of the neurotransmitter, biofouling effects, and the co-existence of interfering agents like ascorbic acid, which are especially common in biological matrices. Techniques to measure dopamine, like liquid chromatography and mass spectrometry are time-consuming, suffer from high costs, and the inability of on-site monitoring, hindering the time-dependent and non-invasive sensing of human midbrain organoid’s dopamine release. In contrast, electrochemical dopamine sensors demonstrate the advantages of low costs, miniaturizability, flexibility, rapid detection, and high sensitivity and selectivity towards dopamine in complex biological samples. In this work, an enzymatic dopamine biosensor was developed to monitor the dopamine release inside the highly complex cell culture supernatant of human midbrain organoids-on-chip over time. Four different working electrodes (a gold-platinum, a gold interdigitated, a gold wire, and a carbon electrode) were modified with the natural biopolymer chitosan, mixed metaloxide nanoparticles, and the enzyme tyrosinase and compared based on their usability, sensitivity, and selectivity to evaluate the best sensor’s performance. The latter two are critical for measuring low dopamine concentrations and avoiding detecting interferences in complex biological samples. The overall best results were observed with the carbon electrode with a limit of detection of 36 nM (n=6) inside a stable buffer and, more importantly, 193nM (n=4) in a complex medium. It was demonstrated that the evaluation of the enzymatic- based carbon electrodes revealed no significant influence of the most common co-existing interference ascorbic acid. The carbon electrode sensor was able to detect and monitor significant differences between healthy and Parkinson’s disease-specific midbrain organoids over an extended cultivation period of seven weeks. Furthermore, the dopamine sensor observed positive effects of dynamic cultivation on organoid differentiation, which was expressed byincreaseddopaminelevelsinthedynamicallyculturedorganoidsupernatants. Additionally, a positive impact was detected after the treatment of patient-specific organoids with a compound. Overall, in the course of the study, an enzymatic carbon-based dopamine sensor with good usability, stability, selectivity, and sensitivity was developed. Furthermore, the practical applicability of the sensor for non-invasive monitoring in organoid-on-chip technology was successfully demonstrated using dynamically cultivated human midbrain organoid-on-chip. This dopamine biosensor demonstrates a possible sensor strategy to integrate into a more expanded drug screening study monitoring the dose-response for personalized Parkinson’s disease models.